1. Field of the Invention
The present invention relates to an immunoassay for measuring the concentration of an object of measurement contained in a specimen by means of an immuno-reagent comprising an antigen or antibody.
2. Description of the Related Art
Heretofore, there have been known various immunoassays making use of an antigen-antibody reaction, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), turbidimetric immunoassay (TIA) and latex agglutination immunoassay (LPIA).
In these immunoassays, the concentration of an object of measurement contained in a specimen is determined from its analytical measurement values (absorbance, transmittance, turbidity, fluorescence, reaction rate and other measured physical values). Calibration for plotting the relationship between the concentration and analytical measurement values of an object of measurement contained in a standard sample is carried out by measuring the standard sample containing the object of measurement in a known concentration in advance so as to determine the concentration of the object of measurement through comparison with the calibration curve. All of these conventional immunoassays are premised on that the specimen and the standard sample are measured in different reaction vessels and that these measurements are always carried out under the same conditions.
However, since there are temperature variations (such as temperatures of apparatus, room, reagent, etc.), variations derived by the stability of the reagent, variations caused by the use state of the reagent (for example, changes in reactivity caused by contamination or the like, the evaporation of water from a reagent bottle, decrease of activity, etc.) in fact, when measurement is newly carried out the following day or several days after the end of a set of continuous measurements or when the lot of reagents is changed, a calibration curve must be newly prepared right before the start of measurement in order to maintain measurement accuracy. That is, in these immunoassays, calibration takes time and it is difficult to make effective use of an immuno-reagent. Even when this calibration is carried out, it is difficult to eliminate an error caused by within-day changes (temperature variations, changes in the intensity of light of a light source, fluctuations in a detector, etc.).
Meanwhile, the standard sample is measured at one point or several points right before the start of measurement to correct the previously prepared calibration curve in order to make effective use of an immuno-reagent.
However, even in this immunoassay, to further improve measurement accuracy, a calibration curve is preferably corrected in advance for each specimen, which is disadvantageous from the viewpoints of time and cost.
Then, an immunoassay for measuring the concentration of an object of measurement effectively and accurately without wasting an immuno-reagent and time has been desired.
To meet this demand, the inventors of the present invention have conducted intensive studies and have found that a reaction between an immuno-reagent and a standard sample containing an object of measurement in a certain concentration (known concentration), and a reaction between the immuno-reagent and a specimen, are carried out in the same reaction vessel continuously to solve the above problem. The present invention has been thus accomplished based on this finding.
That is, a first aspect of the present invention is a method of immunoassay for measuring the concentration of an object of measurement contained in a specimen by means of an immuno-reagent comprising an antigen or antibody for the object of measurement, characterized by comprising: a standard reaction step of reacting a standard sample containing the object of measurement in a certain concentration with the immuno-reagent in a reaction solution; a specimen. reaction step of mixing and reacting the reaction solution of the standard reaction step with the specimen; and a step of determining the concentration of the object of measurement contained in the specimen by comparing the reactivity of the standard reaction step with the reactivity of the specimen reaction step.
A second aspect of the present invention is a method of immunoassay for measuring the concentration of an object of measurement contained in a specimen by means of an immuno-reagent comprising an antigen or antibody for the object of measurement, characterized by comprising: a standard reaction step of reacting a standard sample containing the object of measurement in a certain concentration with an immuno-reagent in a reaction solution; a specimen reaction step of mixing and reacting the reaction solution of the standard reaction step with the specimen; a step of correcting the previously prepared calibration curve using the reactivity of the standard reaction step; and a step of determining the concentration of the object of measurement contained in the specimen from the corrected calibration curve and the reactivity of the specimen reaction step.
A third aspect of the present invention is a method of immunoassay for measuring the concentration of an object of measurement contained in a specimen by means of an immuno-reagent comprising an antigen or antibody for the object of measurement, comprising: two or more standard reaction steps of mixing and reacting two or more standard samples with a reaction solution containing the immuno-reagent sequentially; a specimen reaction step of mixing and reacting the reaction solution of the last standard reaction step with the specimen; a step of preparating a calibration curve from the reactivity of each standard reaction step; and a step of determining the concentration of the object of measurement contained in the specimen by using the calibration curve and the reactivity of the specimen reaction step, wherein at least the standard samples of a second or later standard reaction steps contain the object of measurement in a certain concentration.
A fourth aspect of the present invention is an immunoassay test kit and an immunoassay apparatus used for the above method of immunoassay.